For Dopamine Detection by Immunohisto/cytochemistry. Example for a Rat brain.1. SOLUTIONS TO BE PREPARED - Solution must be prepared as needed.Solution A: 0.1M cacodylate, 10g/L sodium metabisulfite, pH 6.2.Solution B: 0.1M cacodylate, 10g/L sodium metabisulfite, 3-5% glutaraldehyde, pH 7.5.2. RAT PERFUSION - The rat is anaesthetized with sodium pentobarbital or Nembutal and perfused intracardially through the aorta using a pump with Solution A (30 mL): 150-300 mL/min, Solution B (500 mL): 150-300 mL/min.3. POST FIXATION: 15 to 30 minutes in Solution B, then 4 soft washes in 0.05M Tris with 8.5 g/L sodium metabisulfite, pH 7.5 (Solution C).4. TISSUE SECTIONING: Vibratome or cryostat sections can be used.5. REDUCTION STEP: Sections are reduced with Solution C containing 0.1M sodium borohydride for 10 minutes. The sections are washed 4 times in solution C without sodium borohydride.6. APPLICATION OF DOPAMINE ANTIBODY: Use a final dilution of 1:2,500-1:10,000 in Solution C containing 0.1% Triton X100 and 2% non-specific serum. Incubate 12 sections per 2 mL diluted antibody overnight, +4°C. Then wash the sections three times for 10 minutes each in Solution C. (Note that the antibody may be usable at a higher dilution. This should be explored to minimize the possibility of high background. Additionally, note that a change in the buffering system as indicated in the protocol may change the background and antibody recognition). The specific reaction is then revealed by PAP procedure.7. SECOND ANTIBODY: Incubate the sections with a 1:50 to 1:200 dilution of goat anti-rabbit in Solution B containing 1% non-specific serum for either 3 hrs at 20°C or 2 hr at 37°C. Then wash the sections, 3 times, for 10 minutes each with Solution B.8. PAP: Incubate the sections with the appropriate dilution of peroxidase anti-peroxidase (for free floating method) in Solution B containing 1% non-specific serum for 1-2 hours at 37°C. Then wash sections 3 times for 10 min each in solution B.9. VISUALIZATION: The antigen-antibody complexes are visualized using DAB-4-HCl (25 mg/100 mL) (or other chromogen) in 0.05M Tris and filtrated; 0.05% hydrogen peroxide is added. Incubate the sections for 10 minutes at room temp. Stop the reaction by transferring the sections to 5 mL 0.05M Tris. Wash tissue with solution D using 2, 10 min washes. Mount sections on chrome-alum coated slides. Dry overnight at 37°C. Rehydrate sections using conventional histological procedures. Coverslip using rapid mounting media.

Storage

Store undiluted at 2-8°C for one month or (in aliquots) at -20°C for longer.Avoid repeated freezing and thawing.Shelf life: one year from despatch.

Format

Preservatives:

0.005% Merthiolate and 0.05% Sodium Azide

Stabilizers:

50% Glycerol

State:

Liquid purified IgGPurified

Species Reactivity

Species reactivity (tested):

Rat.

Specificity

Specificity:

This antibody recognizes Dopamine. The cross-reactivities were determined using an ELISA test by competition experiments with the following compounds: Compound : Cross-reactivityDopamine-G-BSA: 1L-DOPA-G-BSA: 1/10,000Tyrosine-G-BSA: 1/36,000Tyramine-G-BSA: 1/>50,000Noradrenaline-G-BSA: 1/>50,000Octopamine-G-BSA: 1/>50,000Adrenaline-G-BSA: 1/>50,000Dopamine: 1/>50,000